Neo-Fs Index: a Novel Immunohistochemical Biomarker

Supplementary Materials Neo-fs index: a novel immunohistochemical biomarker panel predicts survival and response to anti-angiogenetic agents in clear cell renal cell carcinoma Jisup Kim, Jee-Young Park, Su-Jin Shin, Beom Jin Lim and Heounjeong Go

Table S1. Univariate analysis to identify a correlation between survival and clinicopathological factors and immunohistochemical results in patients receiving anti-angiogenic therapies

Recurrence-free survival Overall survival (OS) Disease-specific survival (DSS) (RFS) HR (95% CI) p HR (95% CI) p HR (95% CI) p Clinicopathological variables Female (vs Male) 0.874 (0.402-1.899) 0.734 0.874 (0.402-1.899) 0.734 1.049 (0.444-2.479) 0.914 Age ≥55 years 1.171 (0.624-2.199) 0.623 1.171 (0.624-2.199) 0.623 1.334 (0.596-2.985) 0.483 Radical nephrectomy (vs 1.893 (0.675-5.304) 0.225 1.893 (0.675-5.304) 0.225 1.169 (0.502-2.722) 0.717 partial nephrectomy) 3.452 (0.834- 3.452 (0.834- ISUP grade 3‒4 0.087 0.087 2.294 (0.869-6.058) 0.094 14.290) 14.290) Tumor size ≥4 cm 2.618 (1.026-6.679) 0.044 2.618 (1.026-6.679) 0.044 1.739 (0.807-3.749) 0.158 pT3‒4 1.761 (0.807-3.844) 0.156 1.761 (0.807-3.844) 0.156 1.365 (0.649-2.871) 0.411 8.198 (2.323- pN1 (vs pN0/pNx) 3.094 (1.566-6.113) 0.001 3.094 (1.566-6.113) 0.001 0.001 28.932) Lymphovascular invasion 2.541 (1.304-4.953) 0.006 2.541 (1.304-4.953) 0.006 2.307 (1.062-5.012) 0.035 Margin involvement 1.514 (0.634-3.615) 0.350 1.514 (0.634-3.615) 0.350 3.017 (1.146-7.944) 0.025 Necrosis 1.662 (0.796-3.472) 0.176 1.662 (0.796-3.472) 0.176 1.771 (0.865-3.626) 0.118 Sarcomatoid change 2.309 (1.247-4.278) 0.008 2.309 (1.247-4.278) 0.008 1.050 (0.449-2.455) 0.910 mTOR inhibitor recipient 2.781 (1.421-5.444) 0.003 2.781 (1.421-5.444) 0.003 1.869 (0.902-3.872) 0.092 Immunohistochemistry High APC expression 1.549 (0.685-3.505) 0.293 1.549 (0.685-3.505) 0.293 1.589 (0.600-4.210) 0.351 High NOTCH1 expression 1.814 (0.981-3.355) 0.058 1.814 (0.981-3.355) 0.058 1.951 (0.922-4.127) 0.080 10.061 (1.211- 10.061 (1.211- High ARID1A expression 0.033 0.033 NA NA 83.584) 83.584) High FAT1 expression 0.814 (0.289-2.292) 0.697 0.814 (0.289-2.292) 0.697 0.918 (0.274-3.075) 0.890 High VHL expression 0.698 (0.382-1.273) 0.241 0.698 (0.382-1.273) 0.241 1.086 (0.519-2.273) 0.826 High EYS expression 2.718 (1.451-5.092) 0.002 2.718 (1.451-5.092) 0.002 2.848 (1.214-6.679) 0.016 High KMT2D expression 1.577 (0.846-2.940) 0.152 1.577 (0.846-2.940) 0.152 2.102 (0.939-4.707) 0.071 High Filamin A expression 1.928 (0.982-3.786) 0.057 1.928 (0.982-3.786) 0.057 2.213 (0.954-5.132) 0.064 High PTEN expression 0.717 (0.386-1.334) 0.294 0.717 (0.386-1.334) 0.294 1.412 (0.631-3.160) 0.402 High p53 expression 1.363 (0.487-3.818) 0.556 1.363 (0.487-3.818) 0.556 1.543 (0.531-4.480) 0.425

Cancers www.mdpi.com/journal/cancers Cancers 2 of 8

Neo-fs index Log-rank p 0.001 Log-rank p 0.001 Log-rank p 0.096 3.188 (0.837- 0–1 1.953 (0.550-6.934) 0.300 1.953 (0.550-6.934) 0.300 0.089 12.144) 6.678 (2.443- 6.678 (2.443- 3.101 (0.660- 2 <0.001 <0.001 0.152 18.252) 18.252) 14.567) 3 3.370 (1.312-8.655) 0.012 3.370 (1.312-8.655) 0.012 2.739 (0.982-7.641) 0.054 4 2.373 (1.077-5.230) 0.032 2.373 (1.077-5.230) 0.032 2.862 (1.076-7.612) 0.035 5 (reference) 1 - 1 - 1 - p-for trend 0.738 (0.599-0.909) 0.004 0.738 (0.599-0.909) 0.004 0.733 (0.562-0.957) 0.022 Neo-fs index Low (≤4) 1 - 1 - 1 - High (>4) 0.349 (0.177-0.688) 0.002 0.349 (0.177-0.688) 0.002 0.347 (0.157-0.765) 0.009

Neo-fs index: The number of markers with low expression among the five independent prognosticators (APC, NOTCH1, ARID1A, EYS, and Filamin A)

# The number of patients with the Neo-fs index 0–1, 2, 3, 4, and 5 was three, six, nine, 17, and 28, respectively

* mTOR inhibitor, mammalian target of rapamycin (mTOR) inhibitor; CI, confidence interval

Cancers 3 of 8

Table S2. Multivariate analysis to identify a correlation between survival and clinicopathological factors and immunohistochemical results in patients receiving anti-angiogenic therapies

Overall survival (OS) Disease-specific survival (DSS) Recurrence-free survival (RFS)

HR (95% CI) p HR (95% CI) p HR (95% CI) p Clinicopathological variables Tumor size ≥ 4 cm 1.429 (0.516-3.962) 0.492 1.429 (0.516-3.962) 0.492 NA NA pN1 (vs pN0/pNx) 1.702 (0.756-3.836) 0.199 1.702 (0.756-3.836) 0.199 4.681 (1.160-18.894) 0.030 Lymphovascular 1.500 (0.698-3.225) 0.299 1.500 (0.698-3.225) 0.299 1.798 (0.750-4.312) 0.189 invasion Margin involvement NA NA NA NA 2.450 (0.877-6.845) 0.087 Sarcomatoid change 1.496 (0.746-3.000) 0.257 1.496 (0.746-3.000) 0.257 NA NA mTOR inhibitor 1.977 (0.947-4.124) 0.069 1.977 (0.947-4.124) 0.069 NA NA recipient Immunohistochemistry High ARID1A 9.835 (1.100- 9.835 (1.100- 0.041 0.041 2.431 (0.964-6.130) 0.060 expression 87.939) 87.939) High EYS expression 2.433 (1.243-4.762) 0.009 2.433 (1.243-4.762) 0.009 NA NA Neo-fs index 2.987 (0.766- 2.987 (0.766- 0–1 0.115 0.115 0.917 (0.131-6.431) 0.930 11.646) 11.646) 10.806 (2.897- 10.806 (2.897- 2 <0.001 <0.001 0.616 (0.140-2.697) 0.520 40.308) 40.308) 3 3.619 (1.356-9.659) 0.010 3.619 (1.356-9.659) 0.010 0.751 (0.170-3.305) 0.704 4 2.632 (0.995-6.962) 0.051 2.632 (0.995-6.962) 0.051 0.354 (0.086-1.460) 0.151 5 (reference) 1 - 1 - 1 - p-for trend 0.662 (0.516-0.850) 0.001 0.662 (0.516-0.850) 0.001 0.778 (0.573-1.057) 0.108 Neo-fs index Low (≤4) 1 - 1 - 1 - High (>4) 0.314 (0.146-0.679) 0.003 0.314 (0.146-0.679) 0.003 0.473 (0.198-1.132) 0.093

Neo-fs index: The number of markers with low expression among the five independent prognosticators (APC, NOTCH1, ARID1A, EYS, and Filamin A) # The number of patients with the Neo-fs index 0–1, 2, 3, 4, and 5 was three, six, nine, 17, and 28, respectively

* mTOR inhibitor, mammalian target of rapamycin (mTOR) inhibitor; CI, confidence interval.

Cancers 4 of 8

Table S3. Clinicopathological characteristics of the study population based on the Neo-fs index

Clinicopathological Neo-fs index p variables 0–1 (n=12) 2 (n=39) 3 (n=71) 4 (n=140) 5 (n=369) Sex 0.416 Male 9 (75.0%) 32 (82.1%) 57 (80.3%) 99 (70.7%) 277 (75.1%) Female 3 (25.0%) 7 (17.9%) 14 (19.7%) 41 (29.3%) 92 (24.9%) Age (years) 0.460 < 55 years 5 (41.7%) 21 (53.8%) 34 (47.9%) 62 (44.3%) 192 (52.0%) ≥55 years 7 (58.3%) 18 (46.2%) 37 (52.1%) 78 (55.7%) 177 (48.0%) Procedure 0.425 Partial nephrectomy 6 (50.0%) 16 (41.0%) 41 (57.7%) 76 (54.3%) 200 (54.2%) Radical nephrectomy 6 (50.0%) 23 (59.0%) 30 (42.3%) 64 (45.7%) 169 (45.8%) ISUP grade <0.001 1–2 3 (25.0.%) 10 (25.6%) 34 (47.9%) 61 (43.6%) 221 (59.9%) 3–4 9 (75.0%) 29 (74.4%) 37 (52.1%) 79 (56.4%) 148 (40.1%) Tumor size (cm) 0.873 <4 cm 6 (50.0%) 23 (59.0%) 45 (63.4%) 87 (62.1%) 223 (60.4%) ≥4 cm 6 (50.0%) 16 (41.0%) 26 (36.6%) 53 (37.9%) 146 (39.6%) T stage 0.394 pT1–2 7 (58.3%) 29 (74.4%) 58 (81.7%) 109 (77.9%) 290 (78.6%) pT3–4 5 (41.7%) 10 (25.6%) 13 (18.3%) 31 (22.1%) 79 (21.4%) N stage <0.001 pN0/pNx 11 (91.7%) 35 (89.7%) 70 (98.6%) 133 (95.0%) 368 (99.7%) pN1 1 (8.3%) 4 (10.3%) 1 (1.4%) 7 (5.0%) 1 (0.3%) Lymphovascular invasion 0.074 Absent 8 (66.7%) 29 (74.4%) 64 (90.1%) 114 (81.4%) 318 (86.2%) Present 4 (33.3%) 10 (25.6%) 7 (9.9%) 26 (18.6%) 51 (13.8%) Resection margin 0.033 Clear 10 (83.3%) 38 (97.4%) 69 (97.2%) 137 (97.9%) 363 (98.4%) Involved 2 (16.7%) 1 (2.6%) 2 (2.8%) 3 (2.1%) 6 (1.6%) Necrosis 0.093 Absent 8 (66.7%) 32 (82.1%) 59 (83.1%) 116 (82.9%) 318 (86.2%) Present 4 (33.3%) 7 (17.9%) 12 (16.9%) 24 (17.1%) 51 (13.8%) Sarcomatoid change 0.133 Absent 10 (83.3%) 37 (94.9%) 67 (94.4%) 130 (92.9%) 354 (95.9%) Present 2 (16.7%) 2 (5.1%) 4 (5.6%) 10 (7.1%) 15 (4.1%) Anti-angiogenetic agent 0.009 Not received 9 (75.0%) 33 (84.6%) 62 (87.3%) 123 (87.9%) 341 (92.4%) Received 3 (25.0%) 6 (15.4%) 9 (12.7%) 17 (12.1%) 28 (7.6%) mTOR inhibitor 0.016 Not received 10 (83.3%) 35 (89.7%) 65 (91.5%) 131 (93.6%) 353 (95.7%) Received 2 (16.7%) 4 (10.3%) 6 (8.5%) 9 (6.4%) 16 (4.3%)

Cancers 5 of 8

Neo-fs index: The number of markers with low expression among the five independent prognosticators (APC, NOTCH1, ARID1A, EYS, and

Filamin A)

# Of the 638 samples, seven lacked sufficient tumor cells for testing the expression of at least one marker among APC, NOTCH1, ARID1A, EYS,

and Filamin A; these samples were excluded, and 631 samples were analyzed.

* mTOR inhibitor, mammalian target of rapamycin (mTOR) inhibitor

Cancers 6 of 8

Table S4. Immune signature (mean z-scores) in clear cell renal cell carcinoma based on the Neo-fs index

Neo-fs index Immune gene signature p Low (0–1) (mean±SD) High (4–5) (mean±SD) MHC Class I -0.39±0.59 0.15±0.93 <0.001 CD8+ T cells -0.20±0.65 0.05±0.99 0.008 Cytolytic activity -0.18±0.63 0.12±1.06 0.002 pDCs -0.03±0.42 0.09±0.59 0.041 Co-stimulation, APC -0.02±0.37 0.03±0.46 0.305 Co-inhibition, T cell 0.02±1.13 0.06±0.69 0.705 Co-inhibition, APC 0.20±1.97 -0.17±0.52 0.011 Type II IFN Response 0.39±0.75 -0.16±0.55 <0.001

Neo-fs index: The number of markers with low expression among the five independent prognosticators

(APC, NOTCH1, ARID1A, EYS, and Filamin A); SD: standard deviation; pDCs: plasmacytoid dendritic

cells

Table S5. Comparison of mutational landscape in clear cell renal cell carcinoma based on Neo-fs index

Neo-fs index p Total Low (0–2) High (>4)

Total mutation count 6.67±3.53 2.75±2.63 8.63±1.85 0.001

Single nucleotide variant count 5.17±2.89 2.25±2.06 6.63±2.00 0.005

Total indel count 1.33±1.56 0.25±0.50 1.88±1.64 0.030

Frameshift indel count 1.08±1.08 0.50±0.58 1.38±1.19 0.201

Neo-fs index: The number of markers with low expression among the five independent prognosticators (APC,

NOTCH1, ARID1A, EYS, and Filamin A); SD: standard deviation

Cancers 7 of 8

Table S6. The list of genes targeting in the OncoPanel AMC version 4.3

1) Entire exonic sequence were included for detection of Base Substitution, Insertions/Deletions, and Copy Number Alterations ABL1, ABL2, AKT1, AKT2, AKT3, ALK, APC, AR, ARAF, ARID1A, ARID1B, ARID2, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AURKC, AXIN1, AXL, BAP1, BARD1, BRAF, BRCA1, BRCA2, BRD2, BRD3, BRD4, BRIP1, CBFB, CCND1, CCND2, CCND3, CCNE1, CD274, CDH1, CDK12, CDK4, CDK6, CDKN1A, CDKN1B, CDKN2A, DKN2B, CDKN2C, CEBPA, CHEK2, CREBBP, CSF1R, CTNNB1, DDR1, DDR2, DDX3X, DNMT3A, DOT1L, DPYD, EGFR, EPHA3, EPHB4, ERBB2, ERBB3, ERBB4, ERCC2, ERCC4, ERG, ERRFI1, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, EZH2, FAM175A, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FBXW7, FGF19, FGF4, FGFR1, FGFR2, FGFR3, FGFR4, FLCN, FLT1, FLT3, FLT4, FOXL2, FUBP1, GATA2, GEN1, GNA11, GNAQ, GNAS, H3F3A, HDAC9, HGF, HLA-A, HLA-B, HLA-C, HLA-DRB1, HNF1A, HRAS, IDH1, IDH2, IGF1R, IGF2, JAK1, JAK2, JAK3, KDR, KIT, KMT2A, KRAS, LRP1B, LTK, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K4, MAPK1, MAPK3, MAPK8, MCL1, MDM2, MDM4, MED12, MEN1, MET, MITF, MLH1, MPL, MRE11A, MSH2, MSH6, MTAP, MTOR, MYC, MYCN, NBN, NF1, NF2, NFKBIA, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NRAS, NTRK1, NTRK2, NTRK3, NUTM1, PALB2, PARP1, PBRM1, PDGFB, PDGFRA, DGFRB, PIK3CA, PIK3CB, PIK3CD, PIK3R1, PIK3R2, PMS2, POLE, PPARG, PTCH1, PTCH2, PTEN, Genes PTPN11, RAD50, RAD51, RAD51C, RAD51D, RAF1, RARA, RB1, RET, RICTOR, RNF43, ROS1, RSPO1, RSPO2, RUNX1, SDHA, SDHB, SDHC, SDHD, SETD2, SLX4, SMAD2, SMAD4, SMARCA4, SMARCB1, SMO, SOX2, SOX9, SPOP, SRC, STK11, SYK, TERT, TET2, TMPRSS2, TOP1, TOP2A, TP53, TSC1, TSC2, UBE2T, VHL, WT1, XPO1, XRCC2, ZNRF3 2) Partial intronic sequence were included for detection of Rearrangements ALK, EGFR, NTRK1, RET, ROS1, BRAF 3) Hot spot was included for detection of Base Substitution, Insertions/Deletions A1BG, ABCC5, ACVR2A, ADAMTS18, ADNP, AKAP7, AP1S1, ARV1, ASH1L, BAX, CASD1, CBX4, CCDC73, CD3G, CDH26, CEBPZ, CENPV, CKAP2, CLOCK, COBLL1, CPEB2, CRIPAK, DLC1, DNAH12, DOCK3, DPAGT1, DYNC1I2, EBPL, EPPK1, FBXL3, FGFBP1, FMN2, FRG2B, FXR1, GRIN3B, GTPBP2, HIAT1, IMPA1, INO80E, IRS1, KCTD16, KIAA1919, KRT32, LIPT1, MADCAM1, MVK, MYO1A, NIPA2, NNAT, NOS3, NOTCH2NL, NUDT7, OR4M2, PABPC1, PCBP1, PCDHB16, PCMTD1, PREX2, PRIM2, RASA4, RBBP8, RGS12, RHOA, RUFY2, SEC63, SF3B1, SLC23A2, SPRR3, SSTR4, STAMBPL1, STAU2, SULT6B1, SYNJ2, TAS2R19, TAS2R31, TCF7L2, TEAD2, TMEM60, TMPRSS13, TPSD1, WDR55, WDR87, ZFP37, ZNF141, ZNF563, BTK, CBL, IFITM1, IFITM3, KNSTRN, MAX, NFE2L2, PPP2R1A, PTPN11, RAC1, STAT3, U2AF1, KLF4, CIC, GATA3, KIF5B, ADCYAP1, IMPA2, MIR3648-1, DX3X

Cancers 8 of 8

Figure S1. Representative images depicting high and low expression of APC (a), NOTCH1 (b), ARID1A (c), FAT1 (d), VHL (e), EYS (f), KMT2D (g), Filamin A (h), PTEN (i), and p53 (j) in clear cell renal cell carcinoma (x200). APC, NOTCH1, FAT1, EYS, Filamin A, and PTEN were expressed in the cytoplasm, and ARID1A, VHL, KMT2D, and p53 were expressed in the nucleus.

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